The overall objective is the elucidation of mechanisms involved in the regulation of viral gene transcription during viral oncogenesis. Adenovirus 2 will be used as a model virus for studies of viral gene transcription. The specific aims are to elucidate the nature and mechanism of action of the host and viral-coded proteins which regulate (i) the synthesis of specific viral mRNAs by RNA polymerase II during viral replication and in transformed cells, and (ii) the synthesis of discrete low-molecular weight viral RNAs by RNA polymerase III during viral replication. These studies will include: (1) more detailed structural analysis and mapping (with viral DNA restriction endonuclease fragments) of nascent nuclear transcripts, to more clearly define initiation and termination sites; (2) the isolation, via affinity methods, and the subsequent structural and functional analysis of class II and III RNA polymerases and associated factors from both virus-infected and uninfected KB cells; (3) the reconstruction, from purified or partially purified transcription components, of transcription systems in which specific initiation and termination events are manifest; (4) the isolation and structural analysis of functionally active viral templates and specific fragments thereof; (5) the reconstruction of functionally active viral templates from restriction endonuclease fragments of the viral genome and from specific proteins dissociated from intact templates; (6) analysis of the mechanism(s) involved in the presumed induction of structural modifications in the viral template by specific regulatory proteins, including the possible involvement of other cellular components (e.g. enzymes involved in phosphorylation or acetylation; (7) analysis of DNA recognition sites via their functional inactivation with restriction endonucleases. In all these studies the functions of specific components will be inferred on the basis of their abilities to effect specific transcription events analogous to those which occur in vivo. Therefore, the viral RNAs synthesized in the reconstructed transcription systems will be analyzed and compared with in vivo transcripts (insofar as is possible) by a variety of techniques.